Glial reaction is a common feature of neurodegenerative diseases. Recent studies have suggested that reactive astrocytes gain neurotoxic properties, but exactly how reactive astrocytes contribute to ...neurotoxicity remains to be determined. Here, we identify lipocalin 2 (lcn2) as an inducible factor that is secreted by reactive astrocytes and that is selectively toxic to neurons. We show that lcn2 is induced in reactive astrocytes in transgenic rats with neuronal expression of mutant human TAR DNA-binding protein 43 (TDP-43) or RNA-binding protein fused in sarcoma (FUS). Therefore, lcn2 is induced in activated astrocytes in response to neurodegeneration, but its induction is independent of TDP-43 or FUS expression in astrocytes. We found that synthetic lcn2 is cytotoxic to primary neurons in a dose-dependent manner, but is innocuous to astrocytes, microglia, and oligodendrocytes. Lcn2 toxicity is increased in neurons that express a disease gene, such as mutant FUS or TDP-43. Conditioned medium from rat brain slice cultures with neuronal expression of mutant TDP-43 contains abundant lcn2 and is toxic to primary neurons as well as neurons in cultured brain slice from WT rats. Partial depletion of lcn2 by immunoprecipitation reduced conditioned medium-mediated neurotoxicity. Our data indicate that reactive astrocytes secrete lcn2, which is a potent neurotoxic mediator.
TAR DNA binding protein 43 KD (TDP-43) is an essential gene that regulates gene transcription, mRNA splicing and stability. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), ...two fatal neurodegenerative diseases, TDP-43 is fragmented, generating multiple fragments that include the C-terminal fragment of ∼25 KD. The role of these fragments in the pathogenesis of ALS and FTD is not clear. Here we investigated the aggregation propensity in various polypeptide regions of TDP-43 in mammalian cells and the effect of these fragments on cultured neurons. By expressing the full length and various TDP-43 fragments in motor neuron-derived NSC-34 cells and primary neurons, we found that both N- and C-terminal fragments of TDP-43 are prone to aggregate and the C-terminal end of RRM2 region is required, though not sufficient, for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregation with the full-length TDP-43, consequently reducing the nuclear TDP-43. In addition, the TDP-43 fragments can impair neurite growth during neuronal differentiation. Importantly, overexpression of the full-length TDP-43 rescues the neurite growth phenotype whereas knockdown of the endogenous TDP-43 reproduces this phenotype. These results suggest that TDP-43 fragments, particularly the pathologically relevant C-terminal fragments, can impair neuronal differentiation by dominant-negatively interfering with the function of the full length TDP-43, thus playing a role in pathogenesis in ALS and FTD.
Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood–brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, ...transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.
Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood–brain barrier (BBB). Recent studies documented widespread CNS gene transfer after ...intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.
Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other ...neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.
A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally ...eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5′ ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease that causes motor neuron degeneration, progressive motor dysfunction, paralysis, and death. Although multiple causes have been ...identified for this disease, >95% of ALS cases show aggregation of transactive response DNA binding protein (TDP-43) accompanied by its nuclear depletion. Therefore, the TDP-43 pathology may be a converging point in the pathogenesis that originates from various initial triggers. The aggregation is thought to result from TDP-43 misfolding, which could generate cellular toxicity. However, the aggregation as well as the nuclear depletion could also lead to a partial loss of TDP-43 function or TDP-43 dysfunction. To investigate the impact of TDP-43 dysfunction, we generated a transgenic mouse model for a partial loss of TDP-43 function using transgenic RNAi. These mice show ubiquitous transgene expression and TDP-43 knockdown in both the periphery and the central nervous system (CNS). Strikingly, these mice develop progressive neurodegeneration prominently in cortical layer V and spinal ventral horn, motor dysfunction, paralysis, and death. Furthermore, examination of splicing patterns of TDP-43 target genes in human ALS revealed changes consistent with TDP-43 dysfunction. These results suggest that the CNS, particularly motor neurons, possess a heightened vulnerability to TDP-43 dysfunction. Additionally, because TDP-43 knockdown predominantly occur in astrocytes in the spinal cord of these mice, our results suggest that TDP-43 dysfunction in astrocytes is an important driver for motor neuron degeneration and clinical phenotypes of ALS.
•Slow IT injection of rAAV delivers higher transduction to spinal cord compared with the fast injection.•Fast IT injection delivers higher transduction to brain and peripheral tissues compared with ...the slow injection.•Slow IT injection of a therapeutic rAAV led to a slightly better outcome in SOD1-G93A mice compared with the fast injection.•Injection speed should be taken into consideration in future trials of rAAV gene therapy employing IT injection.
Mutant SOD1 causes amyotrophic lateral sclerosis (ALS) by a dominant gain of toxicity. Previous studies have demonstrated therapeutic potential of mutant SOD1-RNAi delivered by intrathecal (IT) injection of recombinant adeno-associated virus (rAAV). However, optimization of delivery is needed to overcome the high degree of variation in the transduction efficiency and therapeutic efficacy. Here, on the basis of our previously defined, efficient IT injection method, we investigated the influence of injection speed on transduction efficiency in the central nervous system (CNS). We demonstrate that slow IT injection results in higher transduction of spinal cord and dorsal root ganglia (DRG) while fast IT injection leads to higher transduction of brain and peripheral organs. To test how these effects influence the outcome of RNAi therapy, we used slow and fast IT injection to deliver rAAVrh10-GFP-amiR-SOD1, a rAAV vector that expresses GFP and an artificial miRNA targeting SOD1, in SOD1-G93A mice. Both slow and fast IT injection produced therapeutic efficacy but the slow injection trended slightly toward a better outcome than the fast injection. These results demonstrate that IT injection speed influences the predominance of gene delivery at different CNS sites and should be taken into consideration in future therapeutic trials involving IT injection.
The Drosophila annotated gene CG5155 encodes a protein that contains 10 Armadillo-repeats and has an unknown function. To fill this gap, we performed loss-of-function studies using RNAi. By analysis ...of four independent Drosophila RNAi lines targeting two non-overlapping regions of the CG5155 transcript, we demonstrate that this gene is required for male fertility. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with the bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 are a subfamily of Armadillo-repeat containing proteins that may have an evolutionarily conserved function in spermatogenesis.
•The Drosophila Armadillo repeat protein Gudu has testis-enriched expression.•Loss of function of Gudu leads to Drosophila male sterility.•Gudu acts in the testis for spermatogenesis.•Gudu is required for the proper function of spermatid individualization complex.
Mitochondria play a pivotal role in many metabolic and apoptotic pathways that regulate the life and death of cells. Accumulating evidence suggests that mitochondrial dysfunction is involved in the ...pathogenesis of amyotrophic lateral sclerosis (ALS). Mitochondrial dysfunction may cause motor neuron death by predisposing them to calcium-mediated excitotoxicity, by increasing generation of reactive oxygen species, and by initiating the intrinsic apoptotic pathway. Morphological and biochemical mitochondrial abnormalities have been described in sporadic human ALS cases, but the implications of these findings in terminally ill individuals or in post-mortem tissues are difficult to decipher. However, remarkable mitochondrial abnormalities have also been identified in transgenic mouse models of familial ALS expressing mutant Cu, Zn superoxide dismutase (SOD1). Detailed studies in these mouse models indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting that mitochondrial dysfunction may be causally involved in the pathogenesis of ALS. Although the mechanisms whereby mutant SOD1 damages mitochondria remain to be fully understood, the finding that a portion of mutant SOD1 is localized in mitochondria, where it forms aberrant aggregates and protein interactions, has opened a number of avenues of investigation. The future challenges are to devise models to better understand the effects of mutant SOD1 in mitochondria and the relative contribution of mitochondrial dysfunction to the pathogenesis of ALS, as well as to identify therapeutic approaches that target mitochondrial dysfunction and its consequences.
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